In this PPG Renewal Application submitted after 2 years of funding, 6 NIH-funded Pi's propose interactive research focusing on the regulation of outward trafficking particulariy to the apical domain of specific transport proteins in polarized epithelial cells from the small intestine, liver, lung and kidney. Four Projects and 3 supporting Cores are proposed. The proteins, each of which contribute to human diseases, are the BB Na/H exchanger NHE3 (Project 1), the intracellular Cl channel CIC-5 (Project 2), ATPase7B (Wilson protein) (Project 3), and CFTR (newly added Project 4). The common questions being addressed are: How is the function of each protein regulated by plasma membrane trafficking or how does the protein regulate plasma trafficking of other transport proteins? What are the nature of the intracellular compartments in which these proteins occur? What proteins do these transporters interact with as part of regulated trafficking? The Cores are an Administrative Core (Core A, Director, M. Donowitz), Cell Culture/Adeno- and Lentivirus Preparation Core (Core B, Director, M. Tse), and Advanced Imaging (Two-Photon Microscopy, Confocal Microscopy, FRET/FRAP) Core (Core C, Director, O. Kovbasnjuk). Project 1, Knockout Models of PDZ Proteins and NHE3 Regulation (PI, M. Donowitz). The hypothesis will be tested that multiple BB PDZ proteins form multiprotein NHE3 complexes which are involved dynamically in NHE3 regulation. BB PDZ protein knockout mice and knockdowns of individual NHERF proteins will be studied in Caco-2 cells to examine the role of these PDZ proteins in NHE3 regulation. Project 2, CIC-5 in Exocytosis and Endocytosis and in NHES/Megalin Trafficking (PI, S. Gugginb). A knockout mouse model of ClC-5 which mimics Dent disease and was shown to have defects in exocytosis as well as endocytosis will be studied for mechanisms of effects on BB trafficking of NHE3, megalin, and NaPi2A. Mechanistic studies of how ClC-5 is involved in regulated exocytosis will examine the role of Rab 11b. Project 3, Cu Homeostasis and ATPaseTB in Polarized Epithelia (PI, A. Hubbard). The routes of trafficking, regulation and mechanisms involved in ATPase7B (Wilson protein) mediated Cu efflux from polarized epithelial will be studied. Associated binding proteins and the role ofthe N- and C-terminus in plasma membrane targeting will be examined. Project 4, Outward trafficking of CFTR (PI, W. Guggino). The processing and outward trafficking of CFTR, its B band, and the mutant A508 will be studied for how the PDZ domain containing proteins CAL and the NHERFs coordinate setting plasma membrane and organellar distribution of CFTR under basal and cAMP stimulated conditions.